Objective To discriminate the species and individual identification with mitochondrial DNA (mtD-NA) sequencing in forensic medicine practice. Methods The multiplex PCR of mtDNA loop - D high - variation region and cytochrome- b region were investigated. The PCR products were detected with silver- stain method,followed by analysis of the PCR products with fluorescence sequence technique. Results The presence of two bands (358bp,279bp ) indicated the samples were from human, while only one band of 358bp indicated nonhuman origin. The part of mitochondrial DNA loop - D high - variation region (15997 ~ 16236) from 131 unrelated individuals of Guangdong population were sequenced. In all of these samples there were 69 nucleotide variations and 67 haplo-types.There was 2.679 mutation sites on average per person. The polymorphism was 97.92% . Conclusion The methods described here are reliable and very useful in species and personal identification of degraded samples.

In a previous study, we cloned popW from Ralstonia solanacearum strain ZJ3721, coding PopW, a new harpin protein. The procaryotically expressed PopW can induce resistance to Tobacco mosaic virus (TMV), enhance growth and improve quality of tobacco, when sprayed onto tobacco leaves. Here, we constructed an expression vector pB- popW by cloning popW into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciens strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens. After screening on MS medium containing kanamycin, PCR and RT-PCR analysis, 21 T3 lines were identified as positive transgenic. Genomic intergration and expression of the transferred gene were determined by PCR and RT-PCR. And GUS staining analysis indicated that the protein expressed in transgenic tobacco was bioactive and exhibited different expression levels among lines. Disease bioassays showed that the transgenic tobacco had enhanced resistance to TMV with biocontrol efficiency up to 54.25%. Transgenic tobacco also exhibited enhanced plant growth, the root length of 15 d old seedlings was 1.7 times longer than that of wild type tobacco. 60 d after transplanting to pots, the height, fresh weight and dry weight of transgenic tobacco were 1.4, 1.7, 1.8 times larger than that of wild type tobacco, respectively.
Agrobacterium tumefaciens ; Animals ; Bacterial Outer Membrane Proteins ; genetics ; Disease Resistance ; genetics ; Genetic Vectors ; Phenotype ; Plant Diseases ; prevention & control ; virology ; Plants, Genetically Modified ; genetics ; Tobacco ; genetics ; Tobacco Mosaic Virus ; Transformation, Genetic

Objective To study etiology screening role of transrectal ultrasonography in male obstructive azoospermia infertility.Methods The clinical data of 328 cases who suspected of being obstructed sperm disease were retrospectively analyzed.TRUS detection was conducted,at the same time,the sperm amount,sperm and semen pH,pure berries quantitative,neutral sugar alpha glycosidase enzymes quantitative,elastic hard protease were tested.Results In 328 cases with male obstructed no sperm,by TRUS detection results,216 cases (65.8％) could find the causes,ejaculatory duct expansion,seminal vesicle gland lesions,prostate midline cyst were the top three causes respectively;112 patients(34.2％) had no obvious abnormal ultrasonic testing.Sperm was not seen in semen of obstructive azoospermia patients and semen pH ＜ 7,pure berries sugar quantitative and quantitative value neutral alpha glycosidase enzymes were very low,hard elastic protease was low.Conclusion The main causes of obstructive azoospermia were ejaculatory duct expansion,seminal vesicle gland lesions,prostate midline cyst,sperm TRUS detection used for diagnosis of high sensitivity,and easy to operate,noninvasive,and combined with seminal plasma biochemical examination,the diagnostic effect is much better.

Objective　To study the relationship between bax gene expression and endometrial carcinoma. Methods　Bax mRNA expression was evaluated by RT-PCR in a series of 8 normal proliferative endometrium (NPE), 6 simple and complex hyperplasia (SCH), 6 atypical hyperplasia (AH) and 42 endometrial carcinoma (EC).Results　All of the 8 NPE showed moderate bax mRNA expression. In 1 of 6 SCH, 2 of 6 AH and 27 of the 42 EC (64.3%), there was overexpresion of bax mRNA. In endometrial carcinoma, with the progression of clinical stage, increase in histologic grade and in depth of invasion to muscle layer, and lymph node metastersis,the positive rate of bax overexpression was correspondingly increased.Conclusion　The results suggest that bax expression affects the balance between the endometrial cell proliferation and apoptosis. Bax might play an important role in carcinogenesis and tumor progression of endometrial carcinoma. As a high risk factor it can be used to evaluate the biological behavior of endometrial carcinoma.

Objective　To study the relationship between bax gene expression and endometrial carcinoma. Methods　Bax mRNA expression was evaluated by RT-PCR in a series of 8 normal proliferative endometrium (NPE), 6 simple and complex hyperplasia (SCH), 6 atypical hyperplasia (AH) and 42 endometrial carcinoma (EC).Results　All of the 8 NPE showed moderate bax mRNA expression. In 1 of 6 SCH, 2 of 6 AH and 27 of the 42 EC (64.3%), there was overexpresion of bax mRNA. In endometrial carcinoma, with the progression of clinical stage, increase in histologic grade and in depth of invasion to muscle layer, and lymph node metastersis,the positive rate of bax overexpression was correspondingly increased.Conclusion　The results suggest that bax expression affects the balance between the endometrial cell proliferation and apoptosis. Bax might play an important role in carcinogenesis and tumor progression of endometrial carcinoma. As a high risk factor it can be used to evaluate the biological behavior of endometrial carcinoma.

OBJECTIVETo develop an effective method for isolating and culturing single cancer stem cells.

METHODSThe capillary glass tube was stretched on fire and connected to a sterile plastic tube to prepare the single cell separation apparatus. Single SW480 cell clone spheres in serum-free culture were marked with CD133 and CK7, and the single cancer stem cells were separated and cultivated in 96-well plates or microdrop covered by paraffin.

RESULTSSW480 cell clone formation rate was about 1.04%, and the cell clone spheres highly expressed CD133 with low CK7 expression. The isolation of the single cancer stem cells showed a success rate of 98.99% using the separation device. The cell division profile was comparable between the cell cultures in microdrop and 96-well plates in the initial 2 cell divisions (P>0.05), whereas prolonged cell division occurred afterwards in the microdrop culture as compared to 96-well plate culture. The cell population expansion of the single cancer stem cells was similar between microdrop culture (11.5%, 22/192) and 96-well plate culture (9.2%, 17/184) (P>0.05).

CONCLUSIONSSingle SW480 cells can develop into cancer stem cell spheres. Microdrop culture is convenient and stable, and can be the primary choice for single cancer stem cell culture.

Through reviewing domestic and foreign literature on augmentative and alternative communication in patients with motor neuron disease，this paper summarized the characteristics of communication disorders in patients with motor neuron disease，the effect caused by poor communication，as well as the application of augmentative and alternative communication. The purpose of this study is to provide an appropriate augmentative and alternative communication system for patients with motor neuron disease with communication disorders，so as to improve the quality of life of patients，improve negative psychological emotions，and reduce the burden of caregivers.

Objective:To analyze the frequency and characteristics of polymyositis (PM) in idiopathic inflammatory myopathy (IIM), and to investigate whether PM is over-diagnosed.Methods:Patients diagnosed as IIM according to the Bohan & Peter criteria of IIM hospitalized in the Department of Rheumatology of China-Japan Friendship Hospital from 2008 to 2019 were involved in the study. Definite PM (dPM) was defined as typical clinical and pathological features including elevated creatine kinase (CK) level, muscle weakness and muscle biopsy findings with endomysial CD8 + T cell infiltration and expression of MHC-1 on sarcolemma. Meanwhile, dermatomyositis (DM), anti-synthase syndrome(ASS), immune-mediated necrotic myopathy(IMNM), sporadic inclusion body myositis(sIBM) and other myopathies were excluded according to the new classification criteria of IIM subtypes respectively. Statistical analysis was performed using SPSS software 24.0. The Kruskal-Wallis test and χ2 test were used to compare the clinical characteristics between the dPM group and other IIM subtypes. Results:A total of 1 259 patients with IIM including 1 015 (80.6%) DM and 244(19.4%) PM were enrolled in this study. According to the strict definition of PM criteria, only 0.5% of patients (6/1 259) in IIM could be diagnosed as dPM. Most PM patients were IMNM and ASS according to the new IIM subtypes criteria, of which 48.0% (117/244) were IMNM and 32.0% (78/244) were ASS. 66.7%(4/6) of dPM patients were women. One complicated with RA, and one was dPM overlaped with systemic sclerosis. All of them had muscle weakness, mild elevation of CK level [611(391,1 451) U/L], and were myositis-specific autoantibodies negative. Except one dPM patients who did not receive immunoregulatory therapy due to chronic obstructive pulmonary disease, the others were administrated with low or medium dose prednisone combined with or without immunosuppressive agents. After a median follow-up of (38±26) months, the muscle strength of dPM patients were improved.Conclusion:dPM is a very rare clinical subtype of IIM. PM is an over-diagnosed entity in clinical practice. Patients with dPM have mild symptoms and good outcome.

Objective:To investigate the level and changing trend of microparticles (MPs) in super-elderly infected patients, and explore its early warning effect on infection.Methods:The infected patients ≥ 85 years old admitted to the Second Medical Center of Chinese PLA General Hospital from December 2018 to March 2019 were selected as the observation group, and the healthy volunteers ≥ 85 years old in the same period were selected as the control group. Venous blood samples were collected at the 2nd hour, the 2nd day and the 7th day after fever, and the inflammatory markers such as white blood cell count (WBC), neutrophil percentage (NEUT), C-reactive protein (CRP) and procalcitonin (PCT) were measured. The levels of MPs were determined by flow cytometry. AnnexinⅤlabeled CD11b positive MPs (AnnexinⅤ +/CD11b + MPs) represented leukocyte microparticles (LMPs), and AnnexinⅤlabeled CD66b positive MPs (AnnexinⅤ +/CD66b + MPs) represented neutrophil microparticle (NMPs). The differences of each index at different time points between the two groups were compared, and the predictive value of each index to the infection of elderly patients was analyzed by receiver operating characteristic (ROC) curve. Results:A total of 38 subjects were enrolled, including 28 cases in the observation group and 10 cases in the control group. The levels of LMPs and NMPs in the observation group increased to the peak at the 2nd hour after fever, and were significantly higher than those in the control group [LMPs (cells/μL): 55.0 (28.8, 197.2) vs. 19.0 (13.5, 28.3), NMPs (cells/μL): 226.5 (123.3, 516.5) vs. 26.5 (22.0, 48.8), both P < 0.01]. With the control of the disease, LMPs and NMPs decreased gradually. The NMPs on the 2nd day was significantly lower than that at the 2nd hour of fever [cells/μL: 106.0 (40.0, 309.0) vs. 226.5 (123.3, 516.5), P < 0.05], and the LMPs and NMPs on the 7th day were significantly lower than those on the 2nd day [LMPs (cells/μL): 17.0 (12.5, 43.8) vs. 42.0 (13.0, 117.0), NMPs (cells/μL): 30.0 (15.8, 62.0) vs. 106.0 (40.0, 309.0), both P < 0.05]. There was no significant difference in the levels of LMPs and NMPs between the two groups on the 7th day. Among the inflammatory markers, the NEUT in the observation group was significantly higher than that in the control group at the 2nd hour of fever (0.70±0.09 vs. 0.59±0.04, P < 0.01), but there was no significant difference in WBC, CRP and PCT between the two groups. On the 2nd day, the inflammatory markers in the observation group reached the peak and were significantly higher than those in the control group [WBC (×10 9/L): 9.33±2.44 vs. 6.37±1.28, NEUT: 0.78±0.08 vs. 0.57±0.04, CRP (mg/L): 5.67±2.99 vs. 0.33±0.18, PCT (μg/L): 0.80±0.67 vs. 0.07±0.03, all P < 0.01]. On the 7th day, the inflammatory markers in the observation group decreased significantly, and there was no significant difference between the observation group and the control group. ROC curve analysis showed that the area under ROC curve (AUC) and 95% confidence interval (95% CI) of LMPs and NMPs on the day of fever were higher than those of WBC, NEUT, CRP and PCT [0.888 (0.763-1.000), 0.973 (0.931-1.000) vs. 0.679 (0.346-0.811), 0.829 (0.700-0.958), 0.607 (0.404-0.811), 0.554 (0.358-0.749)]. Conclusion:LMPs and NMPs are significantly increased in the early stage of fever, which can predict the incidence of infection in the super-elderly patients.

To conduct outbreak identification and transmission factor analysis of typhoid epidemic occurred in Xinqiao town, Jiangyin city from June to September 2016. A total of 14 strains of Salmonella typhi isolated from confirmed cases were collected, and 65 external environment samples and 13 food samples related to the outbreak were taken. Real-time PCR was used to detect specific gene of Salmonella typhi in the samples. Conventional method was used to isolate strains. The strains isolated from both the samples and patients in the epidemic were subjected to antimicrobial susceptibility testing and PFGE molecular characteristics. Salmonella typhi strain was isolated from one external sample (well water of a deli processing plant). The results of drug susceptibility showed that 15 strains were resistant to nalidixic acid. A total of 15 strains of Salmonella typhi were divided into 2 molecular patterns by pulsed field gel electrophoresis. The fingerprints of PFGE from the 13 patients and the environmental isolate were completely consistent, and there was one band difference from the other patient isolate. Combined with the epidemiological investigation and laboratory test results, it was determined that the outbreak was caused by genetic clone of the same Salmonella typhi. Food processing plant should be one of the key links.